A naturally occurring tetrapeptide TKPR (tuftsin, (SEQ ID NO:1) CAS RN=9063-57-4), L-threonyl-L-lysyl-L-prolyl-L-Arginine (SEQ ID NO: 1)
was discovered to stimulate phagocytosis by binding to receptors expressed on the outer surface of neutrophils and macrophages. Phagocytosis constitutes a major line of defense for a host against bacterial infections. Therefore, as a stimulator of phagocytosis, tuftsin would be expected to be a good peptide for imaging sites of infectious inflammation. However studies show that tuftsin labelled with a radionuclide metal undesirably accumulates in non-target tissues.
An alternative approach for imaging infection or inflammation based on the use of a radiolabeled tuftsin receptor antagonist has been disclosed by Pollak A., et al, U.S. Pat. Nos. 5,480,970, 5,659,041, 5,662,885, 5,569,745 and 5,679,642. These patents disclose the use of Tc-99m chelate conjugates of the tuftsin receptor antagonist (see for a review: Nishioka K. et al., Curr. Med. Chem., 1996, 153-66), TKPPR (SEQ ID NO:2), (CAS RN=41961-58-4; or, according to IUPAC nomenclature, L-Arginine, L-threonyl-L-lysyl-L-prolyl-L-prolyl, which has the following structure:
for imaging infection or inflammation. These patents disclose, as chelators, diamidethiols (N2S2) and triamidethiols (N3S). The chelator may be attached to the tuftsin antagonist via a linking group.
Endothelial cells may be defined as an aggregate of cells and/or tissue which may be normal and/or diseased and which may comprise a single layer of flattened transparent endothelial cells that may be joined edge to edge or in an overlapping fashion to form a membrane. Endothelial cells may be found on the free surfaces of the serous membranes, as part of the lining membrane of the heart, blood vessels, and lymphatics, on the surface of the brain and spinal cord, and in the anterior chamber of the eye. Endothelium originates from the embryonic mesoblast and is found associated with heart tissue, including infarcted heart tissue, the cardiovasculature, the peripheral vasculature, such as arteries, veins, and capillaries (the location of which is noted as peripheral to the heart), and the region surrounding atherosclerotic plaques. Additionally, cells that express markers in common with endothelial cells, especially those in contact with the circulation, may also be considered as important targets of the present invention. For instance, melanoma cells that have been observed forming vascular channels and expressing endothelial cell markers as described in A. J. Maniotis et al. (Am. J. Path., 155, 3, 739-752, 1999 and in Science, 285, 5433, 1475, 1999) may be important targets of diagnosis and/or therapy provided by the present invention.
The use of echocardiography for the diagnosis of cardiovascular diseases has generally been limited to indirect methods that involve the detection and quantitation of abnormalities in the wall motion of the heart. Echocardiography has also been used in connection with methods for detecting pathologies of the heart to identify cardiac masses, emboli, thrombi, vegetative lesions (endocarditis), myxomas, and other lesions.
Accordingly, there is a need for improved imaging techniques, including improved contrast agents that are capable of providing medically useful images of the vasculature and vascular-related organs. The imaging techniques, as used herein, include X-ray Imaging, Magnetic Resonance Imaging, Light Imaging, Scintigraphy, and Ultrasound Echograpy.
In particular, as regards ultrasound echography (ultrasound), the quality of images produced from ultrasound has significantly improved in recent years. New imaging methods, especially dedicated or related to contrast agents have been developed, such as, Native Tissue Harmonic Imaging, 2nd Harmonic Imaging, Pulse Inversion Imaging, Acoustically Stimulated Emission (ASE) etc. Nevertheless, further improvements are needed, particularly with respect to images involving tissues that are well perfused with a vascular blood supply.
Accordingly, there is a need for improved ultrasound techniques, including improved contrast agents that are capable of providing medically useful images of the vasculature and vascular-related organs.
The compounds of the present invention may also be useful in the field of angiogenesis. One of ordinary skill will appreciate that a supply of blood vessels is required for tumors to grow beyond a few millimeters in diameter and to metastasize, and that the process by which the blood is provided is generally referred to as angiogenesis. In this process, a vascular supply is developed from existing vasculature for the growth, maturation, and maintenance of tissue. Angiogenesis is a complex multistep process, which involves the endothelial cells of the lumen of blood vessels. Endothelial cells contain all the information necessary to proliferate and migrate to form tubes, branches, and capillary networks.
Targeting angiogenic endothelial cells may be achieved by attaching ligands which will selectively bind to molecules which are upregulated in, on, or near these cells. Such molecules include vascular endothelial growth factor (VEGF) receptors such as Flt-1 (also call VEGFR-1), KDR/Flk-1 (also called VEGFR-2) and NP-1 (also called NRP-1 or neuropilin-1), the αvβ3 and αvβ5 integrins, matrix metalloproteinases, and certain extracellular matrix proteins and fragments thereof VEGF receptors such as NP-1 or KDR are especially attractive targets. VEGF regulates embryonic vasculogenesis as well as physiological and tumor angiogenesis. Mature VEGF is a homodimer in which the monomers are linked “head to tail” by disulfide bridges. A number of VEGF isoforms are produced by alternative splicing from a single gene containing 8 exons. VEGF121 and VEGF165 (containing 121 and 165 amino acids respectively) are the most abundant isoforms. These two VEGF isoforms differ in biological activity. For example, VEGF165 is the stronger endothelial mitogen and binds to heparin, while VEGF121 does not.
The VEGF receptor KDR is one of two VEGF receptor tyrosine kinases (the other being Flt-1) associated primarily with endothelial cells. KDR is present in low amounts in normal mature vessels, but is strongly upregulated at sites of angiogenesis, including angiogenesis induced by hypoxia, inflammation, and cancer. The main site of KDR expression is endothelial cells, but hematopoietic stem cells, megakaryocytes, and retinal progenitor cells also reportedly express it. In addition, some tumor cell lines may express KDR as well
NP-1 is a transmembrane glycoprotein expressed in developing nervous, cardiovascular and skeletal systems as well as in adult endothelial cells, tumor cells and a variety of tissues including placenta, heart, lung, liver, kidney, pancreas, bone marrow stromal cells, osteoblasts and keratinocytes. NP-1 was first identified as being involved in neuronal cell guidance and axonal growth. However, more recently NP-1 was identified as also being a receptor for VEGF165 (and VEGF-B, VEGF-E). Like KDR, NP-1 is strongly upregulated at sites of angiogenesis. NP-1 is a mediator of angiogenesis, particularly in tumors such as breast and prostate carcinoma and melanoma. Cell Vol. 92; 735-74 (1998) Indeed, unlike KDR, NP-1 is abundantly expressed by tumor cells both in vitro and in vivo. Thus, VEGF165 binding to tumor cells is mainly due to NP-1. It has been reported that NP-1 expression in tumors resulted in enlarged tumors associated with substantially increased tumor angiogenesis. Further, it has been suggested that NP-1 retains tumor VEGF and prevents its diffusion out of tumor cells. Miao et al “Neuropilin-1 expression by tumor cells promotes tumor angiogenesis and progression” FASEB J. Vol 14, December 2000.
Thus, molecules specific for VEGF receptors like KDR or, more preferably NP-1, should be valuable in diagnosing, imaging and treating angiogenesis.
Angiogenesis is not only involved in cancer development. Many diseases or conditions affecting different physiological systems include angiogenesis. These include: arthritis and atherosclerotic plaques, which may particularly affect bone and ligaments, diabetic retinopathy, neovascular glaucoma, trachoma and corneal graft neovascularization, which may affect the eye, psoriasis, scleroderma, hemangioma and hypertrophic scarring, which may particularly affect the skin, vascular adhesions and angiofibroma, which may particularly affect the blood system. Therefore, anti-angiogenic factors that work by binding to the afore-mentioned receptors could find a use in the treatment or diagnosis of these diseases and tissues or organs, as well as in cancer therapy and diagnosis.
There is therefore a need for an agent which permits visualization by any of the imaging modalities above cited of endothelial cells, and particularly proliferating and or migrating endothelial cells at sites of angiogenesis. There is a further need for a compound that destroys proliferating endothelial cells at sites of angiogenesis thereby starving the tumor by preventing blood from reaching the tumor or for the treatment of inappropriate angiogenesis in general.